Nitric Oxide Prevents a Pathogen-Permissive Granulocytic Inflammation During Tuberculosis

ARTICLES PUBLISHED: 15 MAY 2017 | VOLUME: 2 | ARTICLE NUMBER: 17072

Nitric oxide prevents a pathogen-permissive granulocytic inﬂammation during tuberculosis Bibhuti B. Mishra1, Rustin R. Lovewell1,AndrewJ.Olive1, Guoliang Zhang2, Wenfei Wang2, Eliseo Eugenin3, Clare M. Smith1,JiaYaoPhuah1, Jarukit E. Long1, Michelle L. Dubuke4, Samantha G. Palace1, Jon D. Goguen1,RichardE.Baker1, Subhalaxmi Nambi1, Rabinarayan Mishra5, Matthew G. Booty1,ChristinaE.Baer1, Scott A. Shaffer4, Veronique Dartois3,BethA.McCormick1, Xinchun Chen2,6* and Christopher M. Sassetti1*

Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen’s environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis risk. These data suggest that M. tuberculosis exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.

espite the ability of Mycobacterium tuberculosis (Mtb)to The primary protective role of NO is anti-inflammatory sustain a persistent infection that can last for decades, most Upon infection with Mtb, mice deficient for inducible NO Dindividuals remain asymptomatic because their immune generation—either due to genetic deletion of the Nos2 gene6 or response effectively contains the pathogen. Only a fraction of chemical inhibition with aminoguanidine (AG)7—suffer from those infected with Mtb will develop the progressive inflammatory high bacterial burdens, weight loss and progressive granulocyte disease, tuberculosis (TB). In these individuals, continual bacterial accumulation that correlates with increased IL-1α and IL-1β replication and progressive necrosis produce cavitary lesions (Supplementary Fig. 1a–e). The granulocytes that accumulate − − contiguous with the airway, which allow bacteria to exit the host in the lungs and spleens of Nos2 / animals were predominantly and infect others1. All infected individuals mount a robust CD11b+, Gr1hi, Ly-6Chi and Ly-6Ghi and had the nuclear immune response to the pathogen, but the immune mechanisms morphology of neutrophils (Fig. 1a and Supplementary Fig. 1f–i). that differentiate protection from disease remain unclear. A smaller proportion of Gr1int myeloid cells with more hetero- Protective immunity to TB requires T cell-derived interferon geneous cytological appearances were also present, as shown in gamma (IFN-γ), which induces the expression of nitric oxide other susceptible mice8–10. Bone marrow chimaeric mice were synthase 2 (Nos2), required for generating nitric oxide (NO) in created to identify the source of protective NO and determine if macrophages. Animals lacking either of these factors suffer this compound’s cell-intrinsic antimicrobial activity is responsible from severe TB disease characterized by high bacterial loads for inhibiting disease. Haematopoietic reconstitution of irradiated − − and granulocytic inflammation2. This correlation between neutro- Nos2 / mice with wild-type, but not Nos2-deficient, bone phils, Mtb burden and pathology is a common feature of both marrow cells inhibited all metrics of disease (weight loss, neutrophil animal models and human TB (refs 3–5). Because NO can kill influx and bacterial replication; Fig. 1b and Supplementary Fig. 2a). Mtb in axenic culture, most models of protective immunity posit To determine if the loss of Nos2 diminished the cell-autonomous that this mediator primarily acts by inhibiting bacterial replication, antimicrobial capacity of the macrophage, wild-type recipients which limits subsequent inflammatory tissue damage. However, were reconstituted with a 1:1 mixture of bone marrow cells from NO also inhibits inflammation by repressing the Caspase1- congenically marked wild-type or mutant mice lacking either dependent processing of pro-interleukin (IL)-1β, and this Nos2 or the IFN-γ receptor (Ifngr). After a month of infection, activity prevents persistent neutrophil recruitment2. We sought to chimaerism of the CD11b+ myeloid compartment was maintained, quantify the individual contribution of these two distinct activities indicating that both genotypes populated the lung and that no obvious of NO. differences in cell death were apparent (Supplementary Fig. 2b,c). At

1Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. 2Guangdong Key Lab of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Guangdong Medical College, Shenzhen 518112, China. 3Public Health Research Institute Center at the International Center for Public Health, New Jersey Medical School – Rutgers, New Jersey 07103, USA. 4Proteomics and Mass Spectrometry Facility, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. 5Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. 6Department of Pathogen Biology, Shenzhen University School of Medicine, Shenzhen 518060, China. *e-mail: [email protected]; [email protected]

NATURE MICROBIOLOGY 2, 17072 (2017) | DOI: 10.1038/nmicrobiol.2017.72 | www.nature.com/naturemicrobiology 1

a b * WT Nos2 KO * P = 0.9773 * 105 105 P = 0.9903 * 5.63 18.2 * * P = 0.6837 ) 104 104 40 0 ) 9 P = 0.9999

P = 0.9811 10 7 * 10 * 3 3

Ly-6G 10 10 ) 6 102 102 30 P = 0.9348 0 0 −10 8 P = 0.7896 P = 0.3699 0 103 104 105 0103 104 105 20 CD11b 6

+ Weight loss (%)

(Gated on CD11b ) C.f.u./lungs (log −20 7 C.f.u./spleen (log 105 105 25.6 64.9 10

104 104 PMNs/lungs (×10 103 103

Ly-6G 0 −30 6 5 102 102 0 0 WT KO KO WT WT KO KO WT WT KO KO WT → → → → → → → → → → → → 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 KO WT>KOKO>KOKO>WT KO KO WT WT KO WT>WT WT WT KO WT WT KO Ly-6C

cde ) 1:1 mix of BM cells ** ** ) **

10 1

10 3 WT (CD45.1) 10 KO (CD45.2) 0 Wild type (WT) Knockout (KO) 2 (CD45.1) (CD45.2) P = 0.9903 −10 3 ** ** 0 1

Reconstitute c.f.u. −20

with mixed BM 10 Weight loss (%) 0 Wild type −30 (CD45.1)

Mtb infection 2 Fold c.f.u. relative to WT (log Fold PMNs relative to WT (log Sort WT and KO −1 −40 −1 cells and determine c.f.u. Normalized log

Knockout cells Knockout (CD45.2) in each population (per 1,000 sorted cells) WT WT WT Wild-type cells (CD45.1) Untreated Untreated Untreated 1 GemcitabineGem+IL-1Rn GemcitabineGem+IL-1Rn GemcitabineGem+IL-1Rn

f Total lung leukocytes WT Ifngr Nos2 Nos2 KO Nos2 KO Nos2 KO 100 Unstained g hi Isotype 80 ** ** WT 25 * * ** 60 Nos2 KO 8 8 P = 0.7029 7 ) ) ) )

40 6 10 10 20 10 20

Percentage of max 7 0 15 0102 103 104 105 7 6 CXCR2 10 5 6 C.f.u./lungs (log C.f.u./lungs (log C.f.u./lungs (log

10 PMNs/lungs (×10 104 5 103 0 6 5 5 102 101 100 Isotype Isotype Isotype CXCR2 MFI (PMNs) Untreated Untreated SCH527123 SCH527123 Anti-Ly-6G Anti-Ly-6G Anti-Ly-6G WT KO Nos2 KO Nos2 KO WT Nos2 KO C3HeB/FeJ Nos2

Figure 1 | Anti-inflammatory activity of Nos2 protects mice from TB disease. a, CD11b+ and Ly6-Ghi (insets in top panels), Ly-6C+ and Ly-6G+ (insets in bottom panels) neutrophils accumulate in the lungs of Mtb-infected Nos2−/− mice (gated on singlet/live cells). b, Bone marrow chimaeric mice were infected with Mtb (notation indicates bone marrow donor genotype → recipient genotype) and lung neutrophils, percentage weight loss and total bacterial burden in the lungs and spleen (expressed in colony-forming units, c.f.u.) were assessed 4 weeks after infection. Values are presented as mean ± s.d. *P <0.05,**P < 0.01, one-way ANOVA with Tukey’s multiple comparison test. c, Schematic for generation of mixed bone marrow chimaeric mice. d, C.f.u. levels were determined in purified haematopoietic cells of indicated genotypes. **P < 0.01, two-way ANOVA with Bonferroni multiple comparison test. e, Mtb infected Nos2−/− mice were treated with gemcitabine (gem), either alone or in combination with IL-1Rn. After 4 weeks of infection, the indicated metrics of disease were quantified. Values (mean ± s.d.) are pooled from two independent experiments. **P < 0.01, one-way ANOVA with Tukey’s multiple comparison test. f, CXCR2 surface expression was determined in CD45+ lung leukocytes (top panel) and mean fluorescence intensity (MFI) of CXCR2 in CD45+ CD11b+ Ly-6G+ F4-80− neutrophils (bottom panel) obtained at 4 weeks post infection. Data are shown as mean ± s.d. g, CXCR1/2 signalling was blocked in infected Nos2−/− mice with SCH-527123 and the number of neutrophils and c.f.u. in the lung were determined after 4 weeks of infection. Values are presented as mean ± s.d. *P < 0.05, two-tailed unpaired t-test with Welch’s correction. h, Bacterial load in the lungs (mean ± s.d.) of anti-Ly-6G- or isotype-treated animals were determined by c.f.u. assay after 14 days of neutrophil depletion. All mice were infected via the aerosol route. **P < 0.001, one-way ANOVA with Tukey’s multiple comparison test. i, Bacterial burden (mean ± s.d.) in the lungs of C3HeB/FeJ mice was determined in the lungs of anti-Ly-6G- or isotype-treated animals after 14 days of neutrophil depletion. **P <0.01,two-tailed unpaired t-test with Welch’s correction. All data shown are representative of at least two independent experiments except in g and i (performed only once). this time point, leukocytes were isolated from the lung, the wild-type and -negative cells recovered from each set of chimaeric animals and mutant cells were separated, and the bacterial burden in each sub- varied to some degree, but the purified cells were found to be an population was quantified. The relative proportion of Ly-6G-positive accurate representation of the populations found in the lung

2 NATURE MICROBIOLOGY 2, 17072 (2017) | DOI: 10.1038/nmicrobiol.2017.72 | www.nature.com/naturemicrobiology

a b e 8 Infected PMNs (lungs) ** ** 0.60 ) ) 12.5 5 100 WT+Mtb 5 WT+Mtb-YFP 0.45 10.0 pdim biosynthesis 80 Nos2 KO+Mtb-YFP 7BL6) mbt cluster 7.5 4 0.30 Carbon source 60 Free radical detoxiﬁcation cells/lung (x10 cells/lung KO/C5 cells/lung (×10 cells/lung 5.0 + + 40 0.15 YFP YFP

2.5 Nos2 Percentage of max

20 0.00 0.0 + − + − −8 −4 4 8 0 Ly-6G Ly-6G Ly-6G Ly-6G Q value 100 101 102 103 104 105 YFP WT Nos2 KO <0.006 −4 c Infected PMNs (spleen) d <0.02 0.025 ** 10 * <0.05 ) 100 ) 5 Relative representation ( representation Relative WT+Mtb 5 0.020 WT+Mtb-YFP 8 80 Nos2 KO+Mtb-YFP 0.015 −8 6 Relative representation (C3HeB/C57BL6) 60 0.010 4 cells/spleen (×10

40 cells/spleen (×10 + 0.005 + 2 Over-represented Under-represented Percentage of max YFP

YFP (BL6susceptible strains) 20 0.000 0 + − + − C3HeB/BL6 0 9 38 32 9147 Nos2 KO/BL6 100 101 102 103 104 105 Ly-6G Ly-6G Ly-6G Ly-6G

YFP WT Nos2 KO

O f g O 0.34x, C3H/B6 (0.007) Mce1 0.30x, Nos2/B6 (0.006) C57BL/6 Hexose Nos2 KO 29.4×, C3H/B6 (0.02)

(log scale) Acetyl CoA C3HeB 66.2×, Nos2/B6 (0.0006) PckA Relative abundance KstR 0.20×, C3H/B6 (0.09) Ratio (C3H/BL6, Nos2 KO/BL6) 0.19×, Nos2/B6 (0.02) TCA

mbtGFEDCBA

Figure 2 | Neutrophilic inflammation produces a growth-permissive environment for Mtb. a, Representative histogram of Mtb-YFP fluorescence in CD11b+Ly-6Ghigh lung leukocytes of wild-type C57BL/6 (WT) or Nos2−/− mice infected with either Mtb or Mtb-YFP via aerosol. b, Enumeration of infected (YFP+) neutrophils (CD11b+Ly-6G+) or monocytes/macrophages (CD11b+Ly-6G−)inWTandNos2−/− mouse lungs after aerosol infection. c, Representative histogram of Mtb-YFP fluorescence in CD11b+Ly-6Ghigh cells. d, Enumeration of infected (YFP+) neutrophils (CD11b+Ly-6G+) or monocytes/macrophages (CD11b+Ly-6G−)inWTandNos2−/− mice spleen after intravenous infection. In b and d,*P <0.05,**P < 0.01, two-tailed unpaired t-test with Welch’s correction. Data are representative of two independent experiments. e, Genome-wide mutant fitness analysis in mouse models of different inflammatory potential. Mice were infected with the Mtb transposon mutant library by intravenous injection. After 4 weeks of selection in mice, the relative abundance of individual Mtb mutants in which a single non-essential gene is disrupted (n = 3,127) was determined using TNseq. Each point represents the relative abundance of mutants lacking an individual gene in Nos2−/− (y axis) or C3HeB mice (x axis), each compared to C57BL/6. Point size indicates statistical significance (log of Q value) between C57BL/6 and Nos2−/− mice. Genes of known function are coloured as described in the panel. Bottom: Venn diagram showing the differential representation of the number of mutants in susceptible strains (Nos2−/− and C3HeB) relative to C57BL6. f, Representative TNseq data for the mycobactin gene cluster. Raw data (upper three tracks) displaying the abundance of each insertion mutant (height of each bar on log scale) in mutant pools selected in the three indicated mouse strains. ‘Relative abundance’ represents the number of Illumina reads corresponding to the insertion site as described in the Methods. The lower track represents the ratio of relative mutant abundance over a window of 20 insertion sites between each mutant mouse and C57BL/6 control (indicated by colour). g, Summary of differential selection on carbon metabolic mutants. The function of each gene or gene cluster is depicted together with the degree of differential abundance and Q valuerelativetotheC57BL/6controlstrain(Q value given in parentheses). In f and g, the colour of each protein represents the sign of its differential representation, with red and green indicating decreased or increased representation in mutant mice, respectively.

(Supplementary Fig. 2d,e). Ifngr-deficient cells harboured significantly infection, when it promotes antibacterial activity in macrophages12 more bacteria than wild type, and no difference in bacterial burden and is essential for the establishment of immunity13.Later,upon − − was observed between wild-type and Nos2 / cells, although both onset of adaptive immunity, IL-1 production is suppressed by NO, − − − − Ifngr / and Nos2 / chimaeric mice had a relatively higher bacterial and over-production of this cytokine can promote neutrophil influx load in their lungs than the wild type (Fig. 1c,d and Supplementary and disease2,14. Thus, to specifically assess the importance of the Fig. 2f,g). It is possible that diffusible NO might partially complement ability of NO to prevent IL-1-dependent neutrophil recruitment, we − − an antimicrobial defect in the Nos2 / cells. However, the greater effect allowed the infection to progress for two weeks before inhibiting of Ifngr deletion indicates that IFN-γ-dependent antimicrobial IL-1 signalling with the IL-1 receptor antagonist (IL-1Rn) and/or processes11 are important for the control of Mtb replication and depleting granulocytic precursors with the cytosine analogue that these processes are largely independent of Nos2. gemcitabine. Gemcitabine treatment reduced the number of lung Because we were unable to detect a cell-autonomous antimicrobial neutrophils, histopathological disease and wasting observed in − − function for Nos2, we hypothesized that the IL-1-modulating anti- Mtb-infected Nos2 / mice and the co-administration of IL-1Rn inflammatory activity of NO might be responsible for protection. accentuated this effect (Fig. 1e). These treatments specifically affected − − IL-1 plays a complex role in TB. This cytokine is produced early in the accumulation of neutrophils in the Nos2 / animals, as the

NATURE MICROBIOLOGY 2, 17072 (2017) | DOI: 10.1038/nmicrobiol.2017.72 | www.nature.com/naturemicrobiology 3

a bcC57BL/6 C57BL/6 + AG *** ** 10 8 * *** ** ***

) 6 6 8 4 6 2 4 0 12-LOX (20×) PMNs/lungs (×10 2 −2 500 μm 500 μm 0

− Fold regulation Alox15 (relative to WT) / −/− − −/− −/− WT +AG +AG WT +AG +AG /− Il1r1 −/− −/− Il1r1− −/− WT+AG Nlrp3 WT+AG Nlrp3 Il1r1 Il1r1 Nlrp3 Nlrp3

Untreated +AG e d 15 *** ** * * ** 8 7 * * 20 *** ) ) ) 6

10 P = 0.3484 P = 0.9997 10 10 P = 0.9969 10 7

5 P = 0.9999 6 C57BL/6 0 500 μm 500 μm 2.0 6 −10 1.5 5 Weight loss (%) C.f.u./lungs (log PMNs/lungs (×10 1.0 5 C.f.u./spleen (log −20 0.5 KO 0.0 −30 4 4 Alox15 WT KO WT KO WT KO WT KO 500 μm 500 μm WT+AG KO+AG WT+AG KO+AG WT+AG KO+AG Alox15 WT+AG KO+AG Alox15 Alox15 Alox15 Alox15 Alox15 Alox15 Alox15

f * g )

P = 0.6292 BALF 6 Parenchyma Bone marrow )

* 15 6 40 20 4

* ) )

6 **

6 ** 16 P = 0.9857 ** 10 P = 0.9966 12 ** 3 30 8 4 5 2 20 2.5 3 2.0 PMNs/BALF (×10

PMNs/lungs (×10 2 1.5 1 10 1.0 1 PMNs/bone marrow (×10

0.5 PMNs/lung parenchyma (×10 0.0 0 0 0

WT WT WT WT WT WT WT KO WT KO WT KO → → → → → → WT+AG KO+AG WT+AG KO+AG WT+AG KO+AG WT KO KO WT KO KO Alox15 Alox15 Alox15 Alox15 KO+12-HETE Alox15 KO+12-HETE Alox15 KO+12-HETE Alox5 Alox15 Alox5 Alox15 KO+AG+12-HETE KO+AG+12-HETE KO+AG+12-HETE Untreated +AG Alox15 Alox15 Alox15 Alox15 Alox15 Alox15

Figure 3 | IL-1-dependent 12/15-LOX products contribute to TB susceptibility in Nos2−/− mice. a, Neutrophil infiltration into the lungs was quantified in each mouse strain 4 weeks after intratracheal infection with ∼1×105 Mtb strain 18b. Aminoguanidine treated groups are indicated (‘AG’). Each symbol represents data from an individual mouse. Data shown as mean ± s.d. b, Relative abundance of Alox15 mRNA in the lungs of animals from a, was quantified by qRT–PCR. In a and b, data (mean ± s.d.) are representative of two independent experiments. *P < 0.05, **P < 0.001, ***P < 0.0001, one-way ANOVA with Tukey’s multiple comparison test. c, WT mice were infected with ∼200 c.f.u. of Mtb H37Rv via the aerosol route. At 4 weeks after infection, 12/15-LOX expression in the lungs was quantified by IHC staining of lung sections of untreated- and AG-treated WT mice. Scale bars, 500 µm. Data are representative of two experiments and, for each condition, three replicates were tested. d, Lung neutrophils, percentage weight loss and total bacterial burden in the lung and spleen were quantified in untreated and AG treated groups of WT and Alox15−/− after 4 weeks of Mtb infection. Data (mean ± s.d.) are representative of six independent experiments. *P <0.05,**P <0.001,***P < 0.0001, one-way ANOVA with Tukey’s multiple comparison test. e, Immunohistochemical staining −/− for anti-Ly-6G in lung sections of Mtb-infected WT and Alox15 mice treated with and without AG. Scale bars, 500 µm. Data are representative of two experiments and for each condition three replicates were tested. f, Lung neutrophils in cohorts of the indicated bone marrow chimaeric mice were determined 5 weeks after infection. Notation indicates bone marrow donor genotype → recipient genotype. Aminoguanidine-treated groups are indicated by ‘AG’. g, Neutrophil numbers in bronchoalveolar lavage fluid (BALF), lung parenchyma after lavage and bone marrow were determined in Mtb-infected WT and Alox15−/− mice, which were treated with 12-HETE and/or AG. In f and g, data (mean ± s.d.) are from one experiment. *P <0.05,**P < 0.001, one-way ANOVA with Tukey’s multiple comparison test. All mice were infected via the aerosol route, except as indicated in a. numbers of other leukocytes were not altered (Supplementary To further investigate the relationship between neutrophils and Fig. 3a). Remarkably, the anti-inflammatory activity of these bacterial replication, we employed more specific reagents to inhibit treatments also restored control of bacterial replication. In this experi- the accumulation of these cells. CXCR2 signalling promotes neutro- ment, the number of neutrophils in the tissue correlated with bacterial phil recruitment to the lungs of Mtb-infected mice15.Weconfirmed − − burden and weight loss, and combination therapy returned these that lung-infiltrating neutrophils expressed CXCR2 and that Nos2 / metrics to a level comparable with wild-type C57BL/6 mice (Fig. 1e mice recruited more CXCR2+ cells (Fig. 1f). Like gemcitabine and and Supplementary Fig. 3b). This observation suggested that sustained IL-1Rn, pharmacological inhibition of CXCR2 reduced the number − − neutrophil recruitment might be the primary cause of both failed of neutrophils and bacteria in the lungs of Nos2 / animals − − anti-mycobacterial immunity and tissue damage in Nos2 / mice. (Fig. 1g). The same effect was observed upon administration of the

4 NATURE MICROBIOLOGY 2, 17072 (2017) | DOI: 10.1038/nmicrobiol.2017.72 | www.nature.com/naturemicrobiology

Table 1 | Distribution of alleles and genotypes for two ALOX12 SNPs between tuberculosis patients (TB) and healthy controls (HC).

SNP ID Trait No. Allele (frequency (%)) Allelic comparison χ2 P P † Del G * adj OR 95% CI rs3840880 TB 943 1,027 (54.5) 859 (45.5) HC 934 1,119 (59.9) 749 (40.1) 11.38 0.0007 0.001 1.25 1.098–1.422 CG rs9904779 TB 943 961 (51.0) 925 (49.0) HC 934 856 (45.8) 1,012 (54.2) 9.89 0.0017 0.027 0.814 0.716–0.925

The genotypes of SNP rs3840880 and rs9904779, which are located at the promoter region of the ALOX12 gene, were determined using the Massarray platform in TB patients (n = 943) and healthy controls (n = 934). The Hardy–Weinberg equilibrium for ALOX12 SNP distribution was analysed in cases and healthy controls. The Pearson ×2 test was used to compare allelic frequencies of SNPs. The unconditional logistic regression adjusted by gender and age was performed to calculate the odd ratios (ORs), 95% confidence intervals (CIs) and corresponding P values with Bonferroni correction. *Compares the difference in allele frequency between TB cases and healthy controls. †P value adjusted by Bonferroni correction for multiple tests. anti-Ly-6G antibody, 1A8, which depleted Gr1hi neutrophils (Fig. 1h spleen, which represented a tractable model for the differential and Supplementary Fig. 3c,d). We conclude that the primary pro- association of Mtb with macrophages versus neutrophils (Fig. 2c,d). tective function of NO in this model was to restrict the accumulation The majority of mutants with significantly altered representation − − of neutrophils, and its direct antimicrobial activity played a relatively in Nos2 / mice displayed comparable differential growth in − − minor role. To extend the observations beyond the Nos2 / mouse C3HeB/FeJ animals (Fig. 2e), indicating that the bacterium is model, we investigated the role of neutrophils in the susceptible encountering a similar environment in these two mouse strains. C3HeB/FeJ mouse. These animals develop inflammatory TB This analysis predominantly identified genes that were less lesions due to increased macrophage necrosis and accumulate a stringently required for growth in the more permissive, hyper- large number of neutrophils in their lungs and spleen, relative to inflammatory hosts (Fig. 2e, green quadrant). The genes of known C57BL/6 mice16 (Supplementary Figs 1h and 3e). As observed in function belonged to a small number of pathways related to stress − − Nos2 / animals, 1A8 administration depleted Gr-1hi neutrophils responses and nutrient acquisition. One class of differentially and reduced lung bacterial burden in C3HeB/FeJ mice (Fig. 1i required genes contained functions implicated in NO resistance − − and Supplementary Fig. 3f), verifying that neutrophil influx that were less stringently required in Nos2 / mice. These included promoted bacterial replication in multiple models of TB susceptibility. the ctpC and sseA genes, which are necessary for toxic radical resistance20,21, the Mpa subunit of the proteasome that influences Neutrophils create growth-permissive environment NO sensitivity22, and genes required for the synthesis of phthiocerol The ability of neutrophil influx to increase the burden of Mtb dimycocerosate (PDIM), a lipid that inhibits the recruitment of suggested that this generally antimicrobial immune response was Nos2-expressing myeloid cells23 (Fig. 2e and Supplementary paradoxically creating a growth-permissive environment for Mtb. Tables 1 and 2). Together, these mechanisms appear to make Mtb To investigate whether a change in the leukocytes encountered by resistant to the levels of NO encountered in vivo. Mtb could contribute to these environmental alterations, mice A second class of pathways related to nutrient acquisition was − − were infected with fluorescent Mtb that could be quantified by found to be less important in Nos2 / and C3HeB/FeJ mice. flow cytometry. We found that the loss of Nos2 altered the Mutants lacking genes required for Mtb’s only iron-scavenging distribution of bacteria between myeloid populations. In both siderophore (mbtGFEADCBA)24 were lost specifically from the lung and spleen, the primary reservoir of Mtb changed from C57BL/6-passaged pool, indicating that scavenging this micro- CD11b+, Ly-6G negative-monocytes/macrophages in wild-type mice nutrient is less important in susceptible mice (Fig. 2e,f). − − to CD11b+,Ly-6Ghi neutrophils in Nos2 / animals (Fig. 2a–d). Neutrophilic inflammation also appeared to alter macronutrient Unlike activated macrophages, neutrophils do not produce availability. Mutations in central metabolic genes and regulators significant amounts of NO (ref. 17) and have a very limited capacity indicated that genes necessary for long-chain fatty acid uptake to restrict the replication of Mtb in vitro18. We confirmed that, (Mce1)25, lipid catabolism (KstR regulon)26 and conversion of − − although wild-type and Nos2 / neutrophils were equally capable lipid substrates to biomass through gluconeogenesis (PckA)27 were − − of killing Gram-negative bacterial pathogens in ex vivo culture, more important for bacterial growth in Nos2 / and C3HeB/FeJ these cells did not alter the viability of Mtb (Supplementary animals (Fig. 2e,g and Supplementary Table 2). We infer that Fig. 4a–c). As a result, we hypothesized that neutrophil-rich Mtb growing in association with neutrophils encounters a more lesions might represent a relatively benign niche for Mtb.To hospitable environment that is replete with micronutrients, such understand how growth in association with neutrophils altered as iron and lipid carbon sources. the bacterial environment, we profiled the behaviour of a bacterial mutant library in mice that develop histiocytic (C57BL/6) and IL-1-dependent 12/15-LOX promotes TB disease − − granulocytic (Nos2 / ) lesions. To assess the impact of altered We hypothesized that the pathological neutrophil recruitment − − host cell association and not simply the loss of NO per se, we also cascade that is derepressed in Nos2 / mice might be a more included the C3HeB/FeJ mouse strain in which neutrophil depletion general mechanism promoting TB susceptibility in some reduces bacterial burden (Fig. 1i). Each mouse was infected with a individuals. Because previous work found that neutrophil influx − − saturated library of ∼50,000 independent Mtb transposon into the lungs of Nos2 / mice required NLRP3 inflammasome- mutants, in which every non-essential gene is disrupted by dependent IL-1 production2, we sought to define the inflammatory insertion. One month after intravenous infection, bacteria were pathway downstream of IL-1 that is responsible for disease in these recovered and the relative abundance of each bacterial mutant in animals. To do this, we profiled the expression of a panel of the three differentially selected libraries was compared by deep proinflammatory mediators in animals that were lacking either sequencing the amplified transposon–chromosome junctions. This Nlrp3 or Il1r1. This panel focused on tumour-necrosis factor approach provides a measure of each gene’s contribution to the (TNF) as a known inflammatory cytokine regulated by IL-1 and pathogen’s fitness under conditions encountered in each mouse the cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, strain19. To maintain the complexity of the library in every which produce potent inflammation-modulating eicosanoids28 animal, we assessed the relative fitness of bacterial mutants in the (Supplementary Fig. 5a). To eliminate the contribution of differential

NATURE MICROBIOLOGY 2, 17072 (2017) | DOI: 10.1038/nmicrobiol.2017.72 | www.nature.com/naturemicrobiology 5

a b rs3840880 rs9904779 4 ** 4 P = 0.9221

2 2 F/R value F/R value

0 0

Mock Mock Allele G Allele G Allele C Allele del

c d e ** ** ** ** 400 ** 400 * 500 ) −1 ) ) −1 −1 ml

5 400

300 300 300 12-HETE (ng ml 12-HETE (ng ml 200

200 200 Neutrophils (10 r = 0.619 100 P < 0.0001

100 100 0 HC LTBI TB Non-TB 0 m 3 m 6 m 12 m 0 2,000 4,000 6,000 8,000 10,000 12-HETE (pg ml−1)

Figure 4 | Increased expression and activity of 12-LOX are associated with active TB in humans. a,b, ALOX12-promoter luciferase reporter plasmids carrying different alleles at rs3840880 or rs9904779 were transfected into HeLa cells. After 48 h, the indicated luciferase activities were quantified. ‘F/R value’ indicates the ratio of Firefly/Renilla luciferase. Values shown as mean ± s.d. **P < 0.01, one-way ANOVA with Tukey’s multiple comparison test. c, Concentrations of 12-HETE in the plasma of healthy controls. HC (n = 10), latent TB infection (LTBI) (n = 10), pulmonary tuberculosis (TB) (n = 20) and subjects suffering from non-TB lung disease, (non-TB) (n = 10) were determined by ELISA. d, 12-HETE levels in the plasma of active TB patients were determined by ELISA at the indicated time points after initiation of anti-TB chemotherapy. ‘m’ represents months of antibiotic treatment. e, 12-HETE levels were significantly correlated with neutrophil counts in the BALF of patients with pulmonary TB, at the time of diagnosis (n = 42). The differences among groups were compared using one-way ANOVA followed by Tukey’s multiple comparison test *P <0.05,**P < 0.01. Pearson’scorrelationcoefficient r and P value of correlation (P) are indicated. bacterial replication in these mice, we used an auxotrophic strain of deletion significantly affected colony forming unit (c.f.u.) numbers Mtb that is unable to replicate during infection, but remains viable in Nos2-sufficient animals, probably because relatively few neutrophils and promotes an inflammatory reaction2,29. As expected, deletion of infiltrate the lung in this situation. Pharmacological blockade of either Nlrp3 or Il1r1 abolished neutrophil recruitment to the lungs 12/15-LOX (ref. 31) had a qualitatively similar effect as genetic of infected animals, even when Nos2 was inhibited (Fig. 3a). Of the deletion of Alox15, ruling out unappreciated developmental effects eicosanoid-biosynthetic enzymes that we profiled, only the expression of the Alox15 mutation (Supplementary Fig. 6c). By these metrics of − − of Alox15, which encodes the 12/15-lipoxygenase (12/15-LOX) disease, Alox15 / mice were essentially resistant to the exacerbated enzyme, correlated with neutrophil influx (Fig. 3b and TB disease caused by Nos2 inhibition (Fig. 3d). Reconstitution of − − − − Supplementary Fig. 5b). Alox15 mRNA levels were significantly wild-type mice with Alox15 / (but not Alox5 / ) bone marrow increased upon Nos2 inhibition, and this induction depended reduced neutrophil influx into the lung. Although the effect of upon both Nlrp3 and the ll1r1. Alox15 protein was predominately Alox15 deletion on neutrophil numbers and c.f.u. was less robust in − − associated with the leukocytic infiltrate in mouse lung and was these chimaeric animals than in intact Alox15 / animals, these data particularly abundant in the context of Nos2 inhibition (Fig. 3c). support an important role for haematopoetically-derived 12/15-LOX − − Consistent with these observations, Alox15 / mice recruited in neutrophil recruitment (Fig. 3f and Supplementary Fig. 6b). very few neutrophils to their lungs upon Mtb infection, when To determine which eicosanoids correlated with 12/15-LOX- compared to wild-type controls. Even Nos2 inhibition did not dependent neutrophil recruitment, we quantified the levels of − − − − promote neutrophil recruitment in Alox15 / animals (Fig. 3d,e). relevant lipid mediators in Alox15 / and wild-type animals in In contrast, deletion of Alox5, which encodes 5-lipoxygenase the presence or absence of Nos2 inhibitor. No significant alterations (5-LOX), had no effect on neutrophil recruitment (Supplementary in COX products were observed between these cohorts. The Fig. 6a). Similarly, mice lacking the leukotriene B4 receptor 1 abundance of both the 5-LOX product (LTB4) and the 12/15-LOX − − (Ltb4r1 / ), which governs neutrophil chemotaxis in response to product (12-hydroxyeicosatetraenoic acid (12-HETE)) correlated 5-LOX-derived LTB4 (ref. 30), had a relatively minor impairment with the degree of inflammation (Supplementary Fig. 7). As both in neutrophil influx when compared to Alox15 deletion of these products are produced by inflammatory leukocytes, we (Supplementary Fig. 6b). As we observed with neutrophil depletion concluded that LOX products are markers of neutrophil influx. − − − − in Nos2 / mice, the reduction in lung neutrophils in Alox15 / However, our genetic data specifically implicated 12/15-LOX mice correlated with a reversal in weight loss, improvement of products as the primary drivers of inflammation. histopathological disease and restoration of bacterial control after 12/15-LOX produces 12-hydroperoxyeicosatetraenoic acid Nos2 inhibition (Fig. 3d,e). Neither neutrophil depletion or Alox15 (12-HpETE) and 12-HETE, which could be responsible for

6 NATURE MICROBIOLOGY 2, 17072 (2017) | DOI: 10.1038/nmicrobiol.2017.72 | www.nature.com/naturemicrobiology

h 80 abeDAPI ALOX12 Merge fMerge Cavities ALOX12 Iba-1 60 MPO cells TNF-α (5)

(4) 20 c MPO d H&E g H&E Levels of expression (a.u.) (3) 0

0 500 1,000 1,500 (2) Distance (μm)

450 μm450 μm (1) 180 μm Caseum Caseum Macro Fibrotic Uninvolved (1) rim (2) rim (3) (4) (5)

Figure 5 | ALOX12 expressed in inflammatory areas of cavitary TB lesions. a,b,c,e, Representative single-channel images of a lung cavity, including the caseous centre at the bottom and adjacent cellular/fibrotic layers, stained for DAPI (a), ALOX12 (b)andMPO(c), and showing a merged image of all three (e). d, H&E staining of the region shown in a,b,c,e. f,Magnification of the area outlined in e, showing co-localization of MPO-positive cells and ALOX12 at the interface between the caseous region and cellular rim. The numbers to the right of e refer to the five regions of the cavitary lesion identified in h,from the caseum and outward into the uninvolved lung tissue. g, H&E staining of the region shown in f.Arrowsinf,g denote MPO- and ALOX12-positive neutrophils with characteristic multilobular nuclei. h, Quantification of ALOX12, TNF-α, Iba-1 and MPO-positive pixels in regions extending from the caseous centre to the outer rim and into the cellular and fibrotic layers, performed in four different cavitary tissues and averaged. Arbitrary units (a.u.) on the y axis indicate the average number of positive pixels in the indicated region. The x axis indicates the ‘distance’ from a fixed point in the caseum. The analysis is described in detail in the Methods. All data are presented as mean ± s.d. pro-inflammatory effects either by acting directly on neutrophils32 association was found for SNPs in genes encoding COX-1 (PTGS1), or through conversion into more potent chemotactic mediators33. COX-2 (PTGS2) or 15-LOX1 (ALOX15). In contrast, among To verify that 12/15-LOX products could contribute to neutrophil 12 SNPs in the 5-LOX gene (ALOX5), one was significantly associ- − − recruitment during Mtb infection, we supplemented Alox15 / ated with TB. More notably, two SNPs (rs3840880 and rs9904779) animals with 12-HETE. This treatment restored neutrophil in the myeloid/platelet-expressed 12-LOX (ALOX12) gene, which numbers in the bronchoalveolar lavage (BAL) to wild-type levels produces 12-HETE in humans, were associated with TB (Table 1). (Fig. 3g). The opposing effects of Nos2 and Alox15 were manifested Both rs3840880 and rs9904779 are located in the promoter predominantly in the airway, and modulation of these mediators region of the ALOX12 gene. Using a luciferase reporter system, had only a modest effect on the number of neutrophils remaining the TB-associated allele of rs3840880 produced significantly in the lung after lavage and no significant effect on the number of higher transcriptional activity than its allelic variant, indicating a CD11b+/Ly-6G+ neutrophil precursors in the bone marrow direct effect of this polymorphism on ALOX12 expression. No (Fig. 3g). 12-HETE supplementation of both wild-type and significant difference in transcription was observed between allelic − − Alox15 / mice increased the total number of lung neutrophils, variants at position rs9904779 (Fig. 4a–b). These results provided and the numbers of these cells correlated with bacterial burden genetic evidence that increased ALOX12 expression could throughout this experiment (Supplementary Fig. 6c). Thus, a promote TB. single 12/15-LOX product was sufficient to promote neutrophil To further investigate whether human 12-LOX activity correlates recruitment to the lung. with inflammatory TB, we assessed the concentration of its product, 12-HETE, in relation to disease, bacterial burden and neutrophil 12-LOX activity is associated with human TB infiltration. The 12-HETE concentration in peripheral blood of − − To understand if the inflammatory pathway delineated in Nos2 / TB patients was significantly higher than in healthy cohorts (HC) mice represents a mechanism of TB susceptibility in natural popu- or patients with non-TB lung diseases (Fig. 4c). Plasma 12-HETE lations, we investigated whether polymorphisms in orthologous levels decreased significantly over the first three months of TB genes were associated with human TB. Significant differences in chemotherapy (Fig. 4d), mirroring the clearance of bacteria. eicosanoid pathways exist between mice and humans. For example, Moreover, in bronchoalveolar lavage fluid (BALF) of TB patients, the 12- and 15-LOX activities possessed by murine 12/15-LOX the level of 12-HETE correlated positively with the abundance of are expressed by separate human proteins. To account for these neutrophils (Fig. 4e). This association was similar to that observed differences, a total of 112 single nucleotide polymorphisms previously for IL-1β and neutrophils in the BALF of TB patients14. (SNPs) in six eicosanoid-modifying genes (Supplementary The association between 12-LOX and granulocytic inflammation Table 3) were genotyped in a cohort of TB patients and healthy was further investigated in human cavitary TB lesions using controls. These SNPs were prioritized based on their minor allele quantitative immunohistochemistry. These lesions consist of an frequency (>10%) and their position in 5′ or 3′ untranslated acellular necrotic centre (caseum), which is sequentially surrounded regions that could alter gene expression. by a myeloid-cell-rich region (‘macrophage rim’) and a fibrotic Polymorphic alleles of the leukotriene A4 hydrolase (LTA4H) capsule. Quantification of multiple lesions indicated that inflamma- gene have previously been associated with TB susceptibility34.In tory markers, such as TNF-α and myeloperoxidase (MPO) expres- general support of these findings, our diseased group was enriched sing neutrophils, were found in the region adjacent to the caseum for the minor alleles of two LTA4H SNPs. However, these (Fig. 5a–g). This region encompassed both the cellular and fibrotic associations did not reach statistical significance after multiple outer edge of the cavity and the macrophage rim. Consistent with testing correction (Supplementary Table 4). Similarly, no significant the pro-inflammatory role played by its murine orthologue, we

NATURE MICROBIOLOGY 2, 17072 (2017) | DOI: 10.1038/nmicrobiol.2017.72 | www.nature.com/naturemicrobiology 7

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. ARTICLES NATURE MICROBIOLOGY found that human 12-LOX expression was concentrated in this under specific pathogen-free conditions and in accordance with the University of TNF-α and MPO-rich region of the cavitary granuloma (Fig. 5h), Massachusetts (UMASS) Medical School, IACUC guidelines. All mouse strains used which was previously defined as an inflammatory region35. in this study were of C57BL/6 background unless otherwise indicated. Collectively, these clinical and histopathological observations Mouse infection. The wild-type strain of M. tuberculosis (Mtb) used in these studies indicate that ALOX12 expression and 12-HETE levels correlate was PDIM-positive H37Rv. Bacteria were cultured in 7H9 medium containing with TB disease, the burden of bacteria and the recruitment of 0.05% Tween 80 and oleic albumin dextrose catalase growth supplement (OADC) fl neutrophils. More generally, these findings support a coordinated enrichment (Becton Dickinson). Bacteria expressing yellow uorescent protein fl (msfYFP) were generated by transformation of H37Rv with plasmid PMV261, role for IL-1 and 12-LOX products in the generation of in ammatory which constitutively expresses msfYFP under the control of the hsp60 promoter. For lesions that promote the replication and dissemination of Mtb. infections, mycobacteria were suspended in phosphate-buffered saline (PBS)-Tween 80 (0.05%); clumps were dissociated by sonication and ∼200 c.f.u. were delivered via Discussion the respiratory route using an aerosol generation device (Glas-Col) or 1 × 106 c.f.u. γ γ by the intravenous route. Mouse infections with the streptomycin auxotrophic strain The protective roles of IFN- and IFN- -inducible NO are often of M. tuberculosis, 18b, were carried out as described previously2. Male mice were attributed to a direct antimicrobial activity, and our data confirm used throughout the study. No statistical criteria were used to determine the mouse that IFN-γ signalling is essential for the control of Mtb intracellular sample sizes. None of the mouse analyses were subject to randomization or blinding. − − growth (Fig. 1d). In contrast, the basis for susceptibility Nos2 / Immunohistochemistry (IHC). Lung tissues were fixed in 10% buffered formalin mice was not related to a cell-autonomous antimicrobial defect. fi fl and embedded in paraf n. Five-micrometre-thick sections were stained with Instead, the primary role of NO was anti-in ammatory, and the haematoxylin and eosin (H&E). All staining was done by the Diabetes and lack of Nos2 increased susceptibility by providing a growth-permissive Endocrinology Research Center histopathology core facility at the University of milieu for the pathogen in association with neutrophils. Because NO Massachusetts Medical School. IHC for 12/15-LOX in the lung sections was done by γ staining the lung sections with 15-LO antibody clone H-235 (sc-32940). Lung cannot account for the observed antimicrobial activity of IFN- −/− signalling, other effector mechanisms induced by this cytokine11 sections from Alox15 mice were used as negative control for these staining. γ must be responsible for the ability of IFN- to control the replication Drug treatment of animals. Aminoguanidine treatment. Mice were supplied with of Mtb. drinking water containing 2.5% aminoguanidine hemisulfate (CAS no. 996-19-0, Neutrophil depletion can extend the survival of Mtb-susceptible Sigma) 7 days before Mtb infection. Water was replaced every week. −/− mouse strains other than Nos2 mice, but the relative contributions −/− fi Gemcitabine treatment. Nos2 mice were treated with gemcitabine hydrochloride of neutrophil-mediated tissue damage versus the ampli cation of (cat. no. G6423, Sigma-Aldrich) via intraperitoneal route, 10 days post Mtb bacterial replication have not been determined in these models. infection. Gemcitabine treatment was repeated every three days until the 25th day As a result, it is currently unclear if the neutrophils in other post infection in a total of four doses of 100 mg per kg per mouse. −/− mouse models are functionally similar to those found in Nos2 –1 + Anakinra treatment. IL-1Rn (Kineret) was given at 25 mg kg via Alzet osmotic mice. Indeed, Ly-6G granulocytes are now understood to encompass pumps, implanted in each animal subcutaneously near the trunk. a variety of subsets that serve both phagocytic and immuno- − − modulatory roles9. Further functional characterization of the 12/15-LOX inhibitors. Baicalein (cat. no. 1761), from TOCRIS, was given to Nos2 / − − – neutrophils that promote Mtb replication in Nos2 / and other mice at a dose of 25 mg kg 1 through Alzet osmotic pumps from 2 days to 28 days mouse strains could provide additional markers of susceptibility post infection. to be investigated in clinical cohorts. 12-HETE treatment. 12-HETE (Cayman Chemicals) was administered to mice via – Although other intracellular pathogens transiently infect Alzet osmotic mini pumps (50 µg kg 1) for four weeks, starting on the day neutrophils during the establishment of infection41 or use these of infection. cells as a primary replication site42, the preferential growth of Mtb CXCR2 inhibitor. CXCR1/2 antagonist SCH527123 (cat. no. CS-0609, ChemScene) – in neutrophil-rich lesions is most reminiscent of Salmonella enterica was administered to animals via Alzet osmotic mini pumps at a dose of 5 mg kg 1, subspecies Typhimurium. This pathogen thrives at sites of for four weeks, beginning one day after infection. inflammation by using oxidized by-products of neutrophil activation as substrates for anaerobic respiration43. Our global Neutrophil depletion. Anti-Ly-6G depleting antibody, clone 1A8 (BioXcell), was phenotypic analysis of bacterial mutants showed no indication of administered as described by Nandi and co-workers46, and 200 µg of 1A8 or isotype altered usage of analogous trimethylamine-N-oxide (TMAO) or control (clone 2A3, BioXcell) antibody was administered to animals via intra- −/− peritoneal injection starting 10 days post-infection and then every other day for nitrate reductases in Nos2 mice (Supplementary Table 1 and 2). 11 days. Ly-6G+ neutrophil depletion was verified by fluorescence-activated cell − Instead, Mtb appears to exploit the increased accessibility of sorting (FACS) analysis of CD11b+ Gr-1+ Ly-6C cell populations, and animals were nutrients in granulocyte-rich lesions (Supplementary Fig. 9). We euthanized at day 24 of infection to determine bacterial burden and cytokines speculate that the inferred abundance of nutrients is due to the in the lungs. cell death observed in advanced TB lesions, which are rich in the Flow cytometry. Single-cell suspensions were prepared from BALF obtained from 44 lipid carbon substrates preferred by Mtb and are likely to contain the mouse lung. Lung tissue was digested with collagenase type IV/DNaseI and iron sources such as haem that Mtb can acquire without mycobac- passed through 40 µm cell strainers to obtain single-cell suspension. Non-specific tin45. A remarkable similarity between these two pathogens is the antibody binding sites were blocked by Fc-Block CD16/32 (clone 93, cat. no. common reliance on inflammation for efficient transmission, for 101319) and the cells were stained with anti-CD3-PE (clone 17A2, cat. no. 100205), fl anti-CD11b-PerCP Cy5.5 (clone M1/70, cat. no. 101227), anti-Ly-6G-FITC (clone Salmonella via the generation of bacteria-rich in ammatory diar- 1A8, cat. no. 127605), anti-Ly-6C-PE (clone HK1.4, cat. no. 128007), anti-Gr1-APC rhoea, and for Mtb via the generation of cavitary lesions that facilitate (clone RB6-8C5, cat. no. 108411), anti-CXCR2/CD182-PE (clone SA04484, cat. the production of infectious aerosols. Enhancement of transmission no. 149303), anti-CD11c-Alexa 700 (clone N418, cat. no. 117319), anti-F4/80-PE-Cy7 provides strong selection for these pathogens to thrive at sites of (clone BM8, cat. no. 123113), anti-CD45-Pacific Blue (clone 30-F11, cat. no. 103126), intense inflammation that would be lethal to other microbes. anti-CD45.1-PE (clone A20, cat. no. 110707) and anti-CD45.2-FITC (clone 104, cat. no. 109804). Antibodies were purchased from Bio Legend. All analyses were performed on live cells only after staining them with fixable live dead stain conjugated Methods with eFluor780, purchased from eBiosciences. For infections with fluorescent H37Rv, − − Mice. C57BL/6 (stock no. 000664), Nos2 / (B6.129P2-Nos2tm1Lau/J, stock lung tissue was prepared as described above and stained with anti-Ly-6G-PacificBlue − − − − no. 002609), Alox15 / (B6.129S2-Alox15tm1Fun/J, stock no. 002778) and Alox5 / (clone 1A8, cat. no. 127612), anti-CD11b-APC-Cy7 (clone M1/70, cat. no. 101226) (B6.129S2-Alox5tm1Fun/J, stock no. 004155), C3HeB/FeJ (stock no. 00658), and live/dead fixable violet dead cell stain (Molecular Probes, cat. no. L-34963) and B6.SJL-Ptprca Pepcb/BoyJ carrying the pan leukocyte marker CD45.1 or Ly-5.1 compared against lung tissue infected with non-fluorescent H37Rv. All the staining − − − − (stock no. 002014), Ltb4r1 / (B6.129S4-Ltb4r1tm1Adl/J, stock no. 008102), Il1r1 / was done according to the manufacturer’s instructions. Lung or spleen cells were − − (B6.129S7-Il1r1tm1Imx/J, stock no. 003245) and Nlrp3 / (B6.129S6-Nlrp3tm1Bhk/J, surface-stained for 30 min at room temperature, and fixed for 20 min at 4 °C using the stock no. 021302) were purchased from the Jackson Laboratory. Mice were housed Cytofix buffer (BD-Biosciences, cat. no. 554655).

8 NATURE MICROBIOLOGY 2, 17072 (2017) | DOI: 10.1038/nmicrobiol.2017.72 | www.nature.com/naturemicrobiology

Cell sorting and morphological analysis. To sort Gr-1high and Gr-1int cells, sonicated. A 0.2 ml volume of single-cell suspension was used to infect different single-cell suspensions were prepared from M. tuberculosis Erdman infected lungs of mouse strains via the lateral tail vein at a concentration of 5 × 106 bacilli per ml. – mice, as described above. After incubating with 5 µg ml 1 Fc-Block (eBiosciences) At indicated times, animals were sacked, and spleen homogenates were plated on – for 15 min at 4 °C, cells were stained with anti-CD11b (clone M1/70) and Gr-1 7H10 plates supplemented with 0.05% Tween 80, OADC and 25 µg ml 1 (clone RB6-8C5), as described above. A live-dead stain was used to exclude dead kanamycin. Colonies were scraped off the plates by a rubber policeman, and cells. Cells were then fixed with Cytofix (BD Biosciences). Fixed cells were further bacterial genomic DNA was prepared as described in ref. 19. Transposon insertion fractionated into CD11b + Gr1high and CD11b + Gr-1int byaFACSAriaIIucellsorter sites were tagged with digital barcodes, amplified, sequenced and quantified as (BD Biosciences). Sorted cells were analysed for purity using FACS Aria, and both the described. To determine the relative abundance, the number of independent Gr-1high and Gr-1int cells were obtained at >90% purity. FACS sorted cells were cytospun transposon–chromosome junctions corresponding to a given gene was divided by onto cytoslides (Thermoscientific), stained with DAPI, and images were acquired in a the number of possible insertion sites in that gene that contained insertions Delta Vision deconvolution microscope. The images were taken with a ×60 objective, (‘Hits/TA’). This metric was compared between samples to determine ‘Relative and the Delta Vision SoftWorx software was used to deconvolve the images. abundance’. Statistical significance was determined using a nonparametric permutation test to calculate a P value and adjusting for multiple testing using the Cytokine measurement and immunoblotting in tissue homogenates. Murine Benjamin–Hochberg correction to obtain the Q value. cytokine concentrations in culture supernatants and cell-free lung homogenates were quantified using commercial enzyme-linked immunosorbent assay (ELISA) qRT–PCR. Total RNA from lung tissue was isolated by homogenizing the lung kits (BD Opt EIA). All samples were normalized for total protein content. tissue in Qiazol (Qiagen) and then using the Qiagen RNeasy Plus universal kit with 12/15-LOX expression in macrophage lysates was determined by western blotting with genomic DNA eliminator solution. Individual RNA samples (100 ng each) were 12-LOX antibody H-100 (sc-32939) purchased from Santa Cruz Biotechnology. subjected to quantitative reverse transcription polymerase chain reaction (qRT–PCR) using the QuantiTect SYBR Green RT–PCR kit. In this method, Generation of bone marrow chimaeric mice. B6.SJL-Ptprca Pepcb/BoyJ mRNA levels for each sample were normalized to glyceraldehyde 3-phosphate (CD45.1/Ly-5.1) mice were lethally irradiated (900–1,000 rads or 90–100 Gy) and dehydrogenase (GAPDH) mRNA levels and then expressed as a fold regulation in reconstituted with a total of 107 donor bone marrow cells from C57BL/6 CD45.2 or gene expression using the SABiosciences RT2 profiler PCR data analysis software − − − − − − Alox15 / , Alox5 / or Nos2 / in CD45.2 background. Mice were allowed to (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). The sequences reconstitute for 8 weeks before performing aerosol infection with H37Rv. of the specific primers are as follows: Approximately 85–95% reconstitution of donor bone marrow was achieved in this experiment. Surface expression of congenic markers CD45.1 and CD45.2 antigens Lta4h: forward: 5′-gctactggcaggtgacaagt-3′ and reverse: 5′-tttccccaaacaaccgtcca-3′ was checked by flow cytometry before (blood) and after (infected lung) infection in Alox5: forward: 5′-ctacgatgtcaccgtggatg-3′ and reverse: 5′-gtgctgcttgaggatgtgaa-3′ each cohort. Ptgs1/Cox1: forward: 5′-cttctccacgatctggcttcgtga-3′ and reverse: 5′- cttgagctgcaggaaatagccact-3′ Mixed bone marrow chimaera generation and cell sorting. Wild-type CD45.1+ Ptgs2/Cox2: forward: 5′-acacactctatcactggcacc-3′ and reverse: 5′- mice were lethally irradiated with two doses of 600 rads. The following day, bone ttcagggagaagcgtttgc-3′ marrow from CD45.1+ wild-type mice and CD45.2+ knockout mice (wild-type, Alox15: forward: 5′-gtctccctgcccgcctggta-3′ and reverse: 5′- − − − − Ifngr / or Nos2 / ) was isolated, red blood cells were lysed using Tris-buffered gggggatccaagggcgtga-3′ ammonium chloride (ACT), and the remaining cells were quantified using a Tnf: forward: 5′-cacagaaagcatgatccgcgacgt-3′ and reverse: 5′- haemocytometer. CD45.1+ and CD45.2+ cells were then mixed equally at a 1:1 ratio cggcagagaggaggttgactttct-3′ and 107 cells from this mixture were injected intravenously into lethally irradiated Gapdh: forward: 5′-aactttggcattgtggaagg-3′ and reverse: 5′- hosts. Reconstitution of host mice lasted 6–8 weeks with preventative sulfatrim acacattgggggtaggaaca-3′. treatment for the first 4 weeks. Mixed bone marrow chimaera mice were then infected by low-dose aerosol with M. tuberculosis H37Rv. Four weeks following Lipid quantification by LC–MS. Lung homogenates were prepared in 50% infection, the lungs of chimaera mice were collected, and single-cell suspensions methanol containing 0.02% bromohydroxytoluene (BHT) by homogenizing the were made following collagenase treatment and tissue disruption. An aliquot of this infected lung samples first in cold PBS + 0.04% BHT, followed by an equal volume of suspension was saved for flow cytometry analysis of the lung population. The 100% chilled methanol + 0.04% BHT. The homogenates were then kept at 4 °C for remaining cells were split equally and stained with either anti-CD45.1 conjugated to 60 min, centrifuged at 4,000 r.p.m. for 20 min at 4 °C and the supernatants were allophycocyanin (APC) or anti-CD45.2 conjugated to phycoerythrin (PE). Stained filtered through 0.2 µm PTFE filters. Extracted lung homogenates were reduced to populations were then incubated with either anti-APC or anti-PE magnetic beads near dryness, resuspended in 1 ml of water, and applied to 3 ml, 500 mg Bond Elute (Miltenyi) following the manufacturer’s instructions. Stained populations were C18 cartridges (Agilent Technologies) pre-conditioned with 10 ml methanol then sorted individually by positive selection using LS-columns following the followed by 10 ml water. The bound material was washed with 4 ml water, 5 ml manufacturer’s instructions (Miltenyi). Following elution of each population, ice-cold hexane and eicosanoids eluted with 5 ml methanol. Samples were then dried purified cells were divided equally and then plated for M. tuberculosis on 7H10 agar by nitrogen and reconstituted in 30 µl of 50% methanol for liquid chromatography– or counted and stained for analysis of cellular purity. Cells from the input mass spectrometry (LC–MS)/MS analysis. homogenate, flow through and the positive sort fractions were stained with fixable Deuterium-labelled or unlabelled internal standards (listed in Supplementary Live/Dead Aqua (Life Tech), anti-CD11b-FITC, anti-CD45.2 PE, anti-CD45.1 APC, Table 5, Cayman Chemical), were suspended in 100% ethanol and serially diluted to anti-CD4 PerCP, Ly6G-Pacific Blue (Biolegend) and analysed on a MacsQuant flow generate an 11-point standard curve. Briefly, each standard solution was added to cytometer (Miltenyi). Samples with >90% were used for subsequent analysis. At 100 µl (10 ng) of the working internal standard solution. The final working – – 21 days after plating, colonies were enumerated and the Mtb levels per sorted cell concentrations of the standard curve ranged from 6.25 ng ml 1 to 10 µg ml 1 for were determined. each eicosanoid. Samples were analysed on a Dionex UltiMate 3000 ultrahigh-performance liquid Isolation of neutrophils from mouse bone marrow. Bone marrow cells were chromatography (UHPLC) apparatus (Thermo Scientific) configured to a TSQ − − isolated from C57BL/6J and Nos2 / mice femurs, red blood cells were lysed using Quantiva (Thermo) triple quadrupole mass spectrometer operating in negative ion – ACT, and single-cell suspension was prepared. Mouse neutrophils were prepared mode. Samples and calibrants were injected (10 µl) at 200 µl min 1 onto a by negative selection using the neutrophil isolation kit from Miltenyi Biotec 100 × 2.1 mm BEH (Waters Corporation) C18 1.7 µm UHPLC column heated at (cat. no. 130-097-658) according to the manufacturer’s protocol. Cell isolation was 40 °C and fitted with a guard column of similar chemistry (Waters). Mobile phase A performed with an LS column. The purity of the neutrophils was tested by was 10 mM aqueous ammonium acetate (pH 5.0) and phase B was 100% acetonitrile. counterstaining the isolated cell population with anti-Ly-6G-APC (clone 1A8, The gradient program was as follows: 0–4 min (20–40% B); 4–14 min (40–80% B); cat. no. 127613) and further analysed in a flow cytometer. Isolated neutrophils were 14–14.1 min (80–95% B); 14.1–16 min (95% B); 16–16.1 min (95–20% B); incubated with Yersinia pestis strains (JG102A with an intact type III secretion 16.1–25 min (20% B). Eicosanoid molecular anions were analysed by scheduled single system and JG102B, which lacks the type III secretion system) and the reaction monitoring (SRM) with parent to fragment ion transitions and optimized Enterohemorrhagic Escherichia coli strain lacking the haemolytic toxin. Cells were collision energies previously determined by direct infusion of eicosanoid standards either activated with phorbol myristate acetate (PMA) or left unactivated during the (Supplementary Table 5). Transitions highlighted in bold were used for quantitative assay. Y. pestis strains were grown in a custom-made serum nutritional medium47. analysis while others were used for secondary confirmation. Mtb Erdman strain was incubated for 16 h with medium alone or with bone marrow Eicosanoids were analysed by generating extracted ion chromatograms from the − − derived neutrophils from wild-type or Nos2 / mice. The percent survival in the quantitative SRM transitions (Supplementary Table 5) and integrating the peak presence of neutrophils was calculated relative to medium control. areas using the Xcalibur (version 3.0.63, Thermo) Quan Browser software module. Chromatograms were background-subtracted (INCOS noise) and smoothed Sample preparation for TnSeq analysis. For infection with Mtb transposon (nine points, Gaussian) before integration. For each analyte, a standard curve – – mutants, bacteria were grown in 7H9 medium containing 0.05% Tween 80, OADC was constructed over the 6.25 ng ml 1 to 10 µg ml 1 range by plotting the ratio –1 and kanamycin (25 µg ml ) to an optical density at 600 nm (OD600) of 1.0. Bacterial of the peak areas of the analyte to a corresponding deuterium-labelled internal cells were pelleted and resuspended in PBS containing 0.05% Tween 80 and standard. Exceptions to this were 11-, 8- and 9-HETE, which were expressed relative

NATURE MICROBIOLOGY 2, 17072 (2017) | DOI: 10.1038/nmicrobiol.2017.72 | www.nature.com/naturemicrobiology 9

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. ARTICLES NATURE MICROBIOLOGY to 12-, 12- and 5-HETE-d8, respectively. Standard curves were fit using a ‘1/x’ antibodies (anti-mouse, anti-goat or anti-rabbit conjugated to Alexa-488, Alexa588 weighting and omitting the origin. Data from study samples were processed in a or Alexa-647) and streptavidin conjugated to Texas Red for mycobacterium similar way to the standards, and eicosanoid concentrations were determined by detection for at least 1 h at room temperature. Tissue sections were mounted using relation to the standard curves. All measured sample concentrations fell within the Prolong Gold Antifade reagent (Invitrogen) with DAPI, and the tissue sections were standard curve with the exception of 12-HETE and PGD2. For the latter analytes, examined in an A1 confocal microscope equipped with spectrum detection and standard curves were extrapolated 5× beyond the top point to allow a unmixing systems (Nikon Instruments). Antibody specificity was confirmed by concentration estimate. replacing the primary antibody with a non-specific myeloma protein of the same isotype or non-immune serum. Human subjects and samples. We used a case–control cohort involving 943 Three-dimensional reconstruction and deconvolution of areas of interest was pulmonary TB patients and 934 healthy controls (HC) from the Shenzhen Third performed from 12 to 25 optical sections obtained at 0.250 or 0.150 µm intervals50. People’s Hospital (previously described in refs 14,48). Diagnosis of active TB was To analyse and quantify the abundance of the study proteins, the number of positive based on clinical symptoms, chest radiography and microscopy for acid fast bacilli pixels as well as their intensity in macrophages were measured in specific regions of (AFB), sputum and/or BALF Mtb culture and response to anti-TB chemotherapy. interest. In addition, for each cell population, a linear intensity histogram was Healthy controls with normal chest radiographic findings and without a clinical generated to examine the expression of each enzyme inside defined cell types or in history of TB were recruited. Mtb-specific interferon gamma release assays (IGRA) caseum. To measure the distance from the centre of the granuloma or cavity in a were used to differentiate individuals with latent TB infection (LTBI) from healthy manner consistent across lesions with caseous foci of varying diameters, we used the controls (HC) without infection as described previously49. All subjects were macrophage layer directly subtending the caseum as the ‘anchor point’ and genetically unrelated members of the Chinese Han population. The ages of each computed the pixel density both in the inward and outward directions from the group (years, mean ± s.d.) were as follows: pulmonary TB, 38.74 ± 12.33; HC, anchor point. To compare protein abundance across tissues, similar numbers of cells 36.46 ± 11.52. The ratio of males to females for the TB and HC groups were 709/234 and areas were included in the three-dimensional deconvoluted optical and 576/358, respectively. All SNPs were in Hardy–Weinberg equilibrium in both reconstructions. This analysis avoids problems associated with inflammatory versus diseased and healthy groups (P > 0.05). normal tissues, where density and structure of the tissue are different. Adjacent Plasma samples were collected from HC (n = 10), LTBI (n = 10), Mtb culture sections were stained with H&E, and the confocal findings were correlated with the confirmed TB (n = 20) and patients with non-TB lung diseases (non-TB, n = 10). histopathology of the same tissue sections. Co-localization and the numbers of The non-TB lung diseases group included patients with pneumonia caused by positive pixels for each colour were quantified by using the imaging software NIS Streptococcus pneumoniae (n = 3), Mycoplasma pneumoniae (n = 2), Elements (Nikon). Analysis of nuclei, macrophage and H&E staining identified at Cytomegalovirus (n = 2) and Klebsiella pneumoniae (n = 3). Whole blood and least five different layers from the centre of the cavity going outward: caseum plasma samples were collected and stored at −80 °C. Unless otherwise indicated, all (acellular); caseum outer rim (interface between caseum and macrophages); samples from patients were collected before initiation of antibiotic treatment. Aliquots macrophage rim (area enriched for Iba-1-positive cells) and uninvolved lung of BALF samples from pulmonary TB patients (n = 42), described previously, were containing minimal infiltration of monocytes and granulocytes. A Student’s t-test used to correlate 12-HETE levels and neutrophil numbers. The levels of 12-HETE in was used to compare fluorescence average intensities in different regions. Individual the BALF and plasma were measured with a kit from Enzo Life Sciences. pixels with an intensity equal to or greater than 8% of the maximum intensity were All protocols involving human samples were approved by the Research Ethics considered positive (P < 0.00042). Committee of Shenzhen Third People’s Hospital, and informed consent was obtained from all participants. The use, for research purposes, of excess BALF left Statistical analysis. The statistical significance of differences between data groups over from clinically indicated bronchoscopies was deemed exempt from a was determined using the unpaired two-tailed Student’s t-test, or one-way requirement for informed consent beyond the consent normally obtained for this ANOVA using Tukey’s multiple comparison test using Graphpad Prism 6 clinical procedure, as previously described. (*P < 0.05, **P < 0.001 and ***P < 0.0001 denote significant differences, compared as indicated in the figures). SNP selection and genotyping. Genomic DNA was prepared from peripheral whole blood according to the standard protocols of the QIAamp DNA Blood Mini kit Data availability. The data that support the findings of this study are available from (Qiagen). SNPs were selected according to methods described previously, with an the corresponding author upon request. additional focus on potential regulatory regions, such as transcription factor binding Received 12 January 2017; accepted 3 April 2017; sites in the promoter region and microRNA target sites in the 3′-untranslated region. SNPs were genotyped using the MassARRAY system (Sequenom), as described published 15 May 2017 elsewhere. References 12-LOX promoter plasmid construction and dual-luciferase assay. A 1,081- 1. Canetti, G. The tubercle bacillus in the pulmonary lesion of man. Am. J. Med. basepair sequence of the 12-LOX promoter region, from −1029 to +52, was cloned Sci. 231, 480 (1956). into the pGL3-Basic vector (Promega) upstream of the firefly luciferase coding 2. Mishra, B. B. et al. Nitric oxide controls the immunopathology of tuberculosis by region. Positive clones were subjected to site-directed mutagenesis using a Quick inhibiting NLRP3 inflammasome-dependent processing of IL-1β. Nat. Change Site-Directed mutagenesis kit (Stratagene) to obtain the desired alleles. Immunol. 14, 52–60 (2013). Transient transfections into HeLa cells were performed using lipofectamine 2000 3. Niazi, M. K. K. et al. Lung necrosis and neutrophils reflect common pathways reagent (Life Technologies). Briefly, 2 × 105 HeLa cells were seeded into 24-well of susceptibility to Mycobacterium tuberculosis in genetically diverse, plates and incubated overnight, the cultured cells were co-transfected with 0.7 µg of immune-competent mice. Dis. Model. Mech. 8, 1141–1153 (2015). constructed vectors, or pGL3-Basic original vector and 0.1 ug of internal control 4. Mattila, J. T., Maiello, P., Sun, T., Via, L. E. & Flynn, J. L. Granzyme B-expressing Renilla luciferase reporter plasmid pRL-CMV. 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M.L.D. and S.A.S. performed LC MS Stroke 37, 3014–3018 (2006). analysis. B.B.M., R.R.L. and C.M.S. analysed the data. B.B.M. and C.M.S. wrote the 32. Reynaud, D. & Pace-Asciak, C. R. 12-HETE and 12-HPETE potently stimulate manuscript and C.M.S. supervised the overall study. intracellular release of calcium in intact human neutrophils. Prostaglandins Leukot. Essent. Fatty Acids 56, 9–12 (1997). Additional information 33. Mrsny, R. J. et al. Identification of hepoxilin A3 in inflammatory events: Supplementary information is available for this paper. a required role in neutrophil migration across intestinal epithelia. Proc. Natl Reprints and permissions information is available at www.nature.com/reprints. – Acad. Sci. USA 101, 7421 7426 (2004). Correspondence and requests for materials should be addressed to X.C. and C.M.S. 34. Tobin, D. M. et al. The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans. Cell 140, 717–730 (2010). How to cite this article: Mishra, B. B. et al. Nitric oxide prevents a pathogen-permissive fl 35. Marakalala, M. J. et al. Inflammatory signaling in human tuberculosis granulocytic in ammation during tuberculosis Nat. Microbiol. 2, 17072 (2017). granulomas is spatially organized. Nat. Med. 22, 531–538 (2016). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in 36. McDonald, B. et al. Intravascular danger signals guide neutrophils to sites of published maps and institutional affiliations. sterile inflammation. Science 330, 362–366 (2010). 37. Hurley, B. P., Pirzai, W., Mumy, K. L., Gronert, K. & McCormick, B. A. Selective Competing interests eicosanoid-generating capacity of cytoplasmic phospholipase A2 in The authors declare no competing financial interests.

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